This proposal seeks to examine in detail the nature of GTP-binding (G) protein activation of the muscarinic potassium (GIRK or KGIRI) channel. It is thought that agonist binding to its transmembrane receptor (R) causes dissociation of the heterotrimeric G protein into its component subunits (Galpha and Gbetagamma) which in turn activate KGIRI channels. Despite intense studies of this system in native membranes, major questions remain. What is the physiological role of differential activation of the channel by one the other or both G-protein subunit components? The answer to this question has not yet been addressed in any system which shows differential regulation by specific G-protein subunits. Cloning of KGIRI channel isoforms has made it feasible to use recombinant components of this system to identify unequivocally the specific effector G-protein subunits as well as the sites in the channel that they act upon. Use of synthetic peptides which affect activation of the channel by specific G-protein subunits can serve as a useful tool in distinguishing physiologically relevant G-protein subunit regulation of the channel. Moreover, structural information derived from such peptides can be used as probes to identify the channel sites of action of specific G-protein subunits. We propose the following two specific aims; (1) Effects of G- protein subunits on native and recombinant KGIRI channels. Recombinant G- protein subunits and KGIRI channels will be coexpressed in Xenopus oocytes and mammalian cell lines. Coexpression of specific G-protein subunits (Gbetagamma or mutant-activated Galpha*) with KGIRI isoforms will test for constitutive activation of these channels. Differential response of KGIRI clones and isoforms to G-protein subunits will be related to their primary structures. Finally, peptides, like QEHA and Galphai 297- 318, will be used as tools to distinguish between activation of Galpha and Gbetagamma subunits in membranes containing native (atrial) or recombinant (oocytes, CHO cells) components of the system. (2) Identification of KGIRI channel regions and amino acid residues responsible for activation by G-protein subunits. The second aim will attempt to identify the regions of KGIRI channels responsible for the activation by G-protein subunits. Crosslinking experiments with peptides shown to affect channel activation by specific G-protein subunits will help localize the relevant regions of interaction. A chimeric approach between two highly related K channels (KGIRIalpha and hpK ATPI), which maybe differentially affected by G-protein subunits, will also be used towards identification of regions responsible for interactions with Gbetagamma or Galpha* subunits. Once the important region(s) have been identified, site-directed mutagenesis will follow to identify the critical amino acid residues of the channel responsible for G- protein subunit activation. Specific amino acid substitutions suggested by the QEHA peptide results will be attempted.